298
M. A. GERENCSER
less produced higher titres with homologous antigens and less non-specific
staining than did conjugates with higher F/P ratios.
IV. REACTIONS OF A C T I N O M Y C E S W I T H F I T C
CONJUGATED ANTISERA
A. Staining procedures
The direct staining method has been used almost exclusively in our
studies of Actinomyces. The antigens consist of cells grown in BHI broth
or on BHI agar slants. The broth cultures are centrifuged, the supernatant
decanted and the cells resuspended in FTA buffer (BBL) to make a barely
turbid suspension. Agar grown cells are washed from the slant and suspended in the same buffer to the same concentration. The addition of a few
glass beads to the tube, followed by vortexing, is helpful in obtaining smooth
suspensions especially of A. israelii. Antigen suspensions may be refrigerated and used for several weeks but the cells do not continue to stain
at maximum intensity indefinitely. For this reason, antigen suspensions
used for control purposes are renewed at least monthly.
Smears are prepared by spreading a small loopful of the suspension on a
circumscribed area of a microscope slide. Smears are allowed to air dry and
are gently heat fixed. One drop of conjugate is placed on each smear and
the slides are incubated for 30 min at room temperature under an inverted
Petri dish containing moist filter paper. Excess conjugate is drained off and
the slides washed in phosphate buffered saline p H 7.2 (FTA Buffer, BBL),
for 10 min, counterstained with Evans Blue (1 .O% aqueous) for 5 rnin and
finally rinsed for 1 min each, in two changes of carbonate buffer, p H 9.0.
Alternatively, smears are counterstained with Bovine Albumin Rhodamine Labelled Counterstain (BBL). I n this case, the rhodamine counterstain is added to each conjugate before it is applied to the smears and
washing is carried out by placing the slides in two changes of pH 7.2
phosphate buffered saline for 5 min. each, followed by two changes of
carbonate buffer, pH 9.0, 1 min each. The rhodamine albumin counterstain has been used most often on direct smears from dental plaque,
faeces and other clinical material.
Slides are mounted in buffered glycerol saline, p H 9.0, and examined
under a suitable microscope. Equipment for examination of FA slides will
not be discussed here as such information is available in a number of
publications (Nairn, 1964; Goldman, 1968; Johnson, 1970; Cherry, 1974).
Originally, we used a Leitz Ortholux microscope equipped with an HBO
200 mercury lamp, cardioid darkfield condenser, BG-12 primary filter and
an OG-1 eyepiece filter. Now we are using the same microscope with an
M. A. GERENCSER
less produced higher titres with homologous antigens and less non-specific
staining than did conjugates with higher F/P ratios.
IV. REACTIONS OF A C T I N O M Y C E S W I T H F I T C
CONJUGATED ANTISERA
A. Staining procedures
The direct staining method has been used almost exclusively in our
studies of Actinomyces. The antigens consist of cells grown in BHI broth
or on BHI agar slants. The broth cultures are centrifuged, the supernatant
decanted and the cells resuspended in FTA buffer (BBL) to make a barely
turbid suspension. Agar grown cells are washed from the slant and suspended in the same buffer to the same concentration. The addition of a few
glass beads to the tube, followed by vortexing, is helpful in obtaining smooth
suspensions especially of A. israelii. Antigen suspensions may be refrigerated and used for several weeks but the cells do not continue to stain
at maximum intensity indefinitely. For this reason, antigen suspensions
used for control purposes are renewed at least monthly.
Smears are prepared by spreading a small loopful of the suspension on a
circumscribed area of a microscope slide. Smears are allowed to air dry and
are gently heat fixed. One drop of conjugate is placed on each smear and
the slides are incubated for 30 min at room temperature under an inverted
Petri dish containing moist filter paper. Excess conjugate is drained off and
the slides washed in phosphate buffered saline p H 7.2 (FTA Buffer, BBL),
for 10 min, counterstained with Evans Blue (1 .O% aqueous) for 5 rnin and
finally rinsed for 1 min each, in two changes of carbonate buffer, p H 9.0.
Alternatively, smears are counterstained with Bovine Albumin Rhodamine Labelled Counterstain (BBL). I n this case, the rhodamine counterstain is added to each conjugate before it is applied to the smears and
washing is carried out by placing the slides in two changes of pH 7.2
phosphate buffered saline for 5 min. each, followed by two changes of
carbonate buffer, pH 9.0, 1 min each. The rhodamine albumin counterstain has been used most often on direct smears from dental plaque,
faeces and other clinical material.
Slides are mounted in buffered glycerol saline, p H 9.0, and examined
under a suitable microscope. Equipment for examination of FA slides will
not be discussed here as such information is available in a number of
publications (Nairn, 1964; Goldman, 1968; Johnson, 1970; Cherry, 1974).
Originally, we used a Leitz Ortholux microscope equipped with an HBO
200 mercury lamp, cardioid darkfield condenser, BG-12 primary filter and
an OG-1 eyepiece filter. Now we are using the same microscope with an